Pre-visualization of the normalized count data before any differential analysis

Load

Libraries and functions

Warning message in is.na(x[[i]]):
“is.na() applied to non-(list or vector) of type 'environment'”
==========================================================================
*
*  Package WGCNA 1.63 loaded.
*
*    Important note: It appears that your system supports multi-threading,
*    but it is not enabled within WGCNA in R. 
*    To allow multi-threading within WGCNA with all available cores, use 
*
*          allowWGCNAThreads()
*
*    within R. Use disableWGCNAThreads() to disable threading if necessary.
*    Alternatively, set the following environment variable on your system:
*
*          ALLOW_WGCNA_THREADS=<number_of_processors>
*
*    for example 
*
*          ALLOW_WGCNA_THREADS=4
*
*    To set the environment variable in linux bash shell, type 
*
*           export ALLOW_WGCNA_THREADS=4
*
*     before running R. Other operating systems or shells will
*     have a similar command to achieve the same aim.
*
==========================================================================


Allowing multi-threading with up to 4 threads.
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."

Data

GF_104w_F_1_2GF_104w_F_2_2GF_104w_F_3_2GF_104w_M_1_2GF_104w_M_2_2GF_52w_M_1_2GF_52w_M_2_2GF_52w_M_3_2GF_52w_M_4_2GF_8w_M_1_2SPF_104w_M_14_2SPF_8w_F_1_2SPF_8w_F_3_2SPF_8w_F_4_2SPF_8w_F_5_2GF_8w_F_1_2GF_8w_F_2_2GF_8w_F_3_2GF_8w_F_4_2GF_8w_F_5_2
0610005C13Rik 0.0000 0.00000 0.0000 1.768593 1.259244 0.8418979 4.942785 5.603216 7.231584 8.47277 5.28524 3.500355 2.112002 2.602398 0.00000 18.24998 0.00000 2.025447 5.268672 0.00000
Erg28 868.4015 749.64927 910.3535 886.064952 992.913601 508.5063502530.855142 514.375197 738.344705 603.98749 692.36648 763.077332 744.480801 908.237050 957.79030 898.54336 682.26034 612.697796 593.252445 809.27852
0610009B22Rik1021.3276 1169.19730 1180.9991 1224.750457 1558.313990 1038.9020466876.850113 1016.423320 877.914273 948.95029 1206.79653 1268.295198 1164.769254 1295.994415 1446.89758 1040.24912 1020.67017 1142.352254 959.952003 1094.00091
0610009L18Rik 28.2185 36.20465 18.2253 20.338816 38.406931 31.9921214 26.691041 22.412863 4.338950 61.73018 45.80542 42.004257 45.408049 102.794738 72.02239 48.30878 19.58642 11.139960 45.310578 24.36813
0610009O20Rik 217.5555 278.98879 266.0893 226.379868 271.996624 299.7156634349.949200 395.587025 309.511786 360.69794 178.81730 274.194454 252.384272 227.709862 184.89330 231.88216 158.86764 271.409933 304.529230 269.33199
0610010B08Rik 0.0000 0.00000 0.0000 0.000000 0.000000 0.0000000 0.000000 0.000000 0.000000 0.00000 0.00000 0.000000 0.000000 0.000000 0.00000 0.00000 0.00000 0.000000 0.000000 0.00000

Sample clustering

With all genes

Without X/Y genes

Gene co-expression network analysis

Build network

  1. 24421
  2. 60
  1. 10219
  2. 60
pickSoftThreshold: will use block size 4378.
 pickSoftThreshold: calculating connectivity for given powers...
   ..working on genes 1 through 4378 of 10219
   ..working on genes 4379 through 8756 of 10219
   ..working on genes 8757 through 10219 of 10219
   Power SFT.R.sq  slope truncated.R.sq  mean.k. median.k.  max.k.
1      2    0.144 -0.617          0.901 644.0000  6.20e+02 1390.00
2      4    0.691 -1.460          0.964 114.0000  9.21e+01  438.00
3      6    0.783 -1.840          0.970  31.1000  1.94e+01  198.00
4      8    0.790 -2.020          0.962  11.0000  5.03e+00  106.00
5     10    0.832 -1.990          0.985   4.6100  1.49e+00   63.10
6     12    0.851 -1.960          0.988   2.1900  4.90e-01   40.10
7     14    0.876 -1.890          0.988   1.1500  1.72e-01   26.70
8     16    0.886 -1.820          0.971   0.6430  6.43e-02   18.40
9     18    0.899 -1.780          0.970   0.3830  2.50e-02   13.60
10    20    0.882 -1.770          0.946   0.2400  1.02e-02   10.40
11    22    0.917 -1.720          0.984   0.1560  4.29e-03    8.11
12    24    0.902 -1.700          0.962   0.1050  1.86e-03    6.46
13    26    0.408 -2.230          0.416   0.0724  8.32e-04    5.23
14    28    0.416 -2.190          0.427   0.0514  3.76e-04    4.28
15    30    0.931 -1.630          0.984   0.0372  1.74e-04    3.55

Parameters for network construction

  • Soft-thresholding power parameter: 16
  • Minimum module size: 25
  • Height for module merging: 0.2 (below)
  • Biweight Midcorrelation
ERROR while rich displaying an object: Error in sprintf(wrap, header, body): 'fmt' length exceeds maximal format length 8192

Traceback:
1. FUN(X[[i]], ...)
2. tryCatch(withCallingHandlers({
 .     rpr <- mime2repr[[mime]](obj)
 .     if (is.null(rpr)) 
 .         return(NULL)
 .     prepare_content(is.raw(rpr), rpr)
 . }, error = error_handler), error = outer_handler)
3. tryCatchList(expr, classes, parentenv, handlers)
4. tryCatchOne(expr, names, parentenv, handlers[[1L]])
5. doTryCatch(return(expr), name, parentenv, handler)
6. withCallingHandlers({
 .     rpr <- mime2repr[[mime]](obj)
 .     if (is.null(rpr)) 
 .         return(NULL)
 .     prepare_content(is.raw(rpr), rpr)
 . }, error = error_handler)
7. mime2repr[[mime]](obj)
8. repr_latex.matrix(obj)
9. repr_matrix_generic(obj, sprintf("\\begin{tabular}{%s}\n%%s%%s\\end{tabular}\n", 
 .     cols), "%s\\\\\n\\hline\n", "  &", " %s &", "%s", "\t%s\\\\\n", 
 .     "%s &", " %s &", escape_fun = latex_escape_vec, ...)
10. sprintf(wrap, header, body)
Erg280610009B22Rik0610009L18Rik0610009O20Rik0610010F05Rik0610010K14RikMettl260610012G03Rik0610030E20Rik0610037L13RikZw10ZwintZxdaZxdbZxdcZyg11bZyxZzef1Zzz3Hikeshi
GF_104w_F_1_2868.4015 1021.328 28.21850 217.5555 411.4439 1351.757 283.0953 693.6289 899.3508449.6754 618.0761 1471.913 297.6596 130.1692 923.92821333.552 2864.633 396.8795 752.7967668.1412
GF_104w_F_2_2749.6493 1169.197 36.20465 278.9888 461.0769 1516.336 207.6443 686.8236 759.2329526.0323 797.5672 1539.763 243.8490 198.0607 981.78501340.637 3358.514 364.1762 597.3768703.8610
GF_104w_F_3_2910.3535 1180.999 18.22530 266.0893 288.8709 1852.601 222.3486 868.4353 540.3800444.6972 731.7456 1375.099 153.0925 171.3178 922.19991181.910 3058.205 209.5909 644.2642770.9300
GF_104w_M_1_2886.0650 1224.750 20.33882 226.3799 278.5534 1551.056 190.1237 823.2799 525.2720607.5116 466.9085 1752.675 172.4378 344.8756 683.56111295.494 2789.955 261.7517 944.4285687.9826
GF_104w_M_2_2992.9136 1558.314 38.40693 271.9966 304.1073 1911.532 243.0340 778.8422 707.6949586.8075 878.3224 1957.494 147.9611 249.3302 1003.61721205.726 2819.446 200.8494 785.1384583.6594
GF_52w_M_1_2508.5064 1038.902 31.99212 299.7157 392.3244 1173.606 182.6919 589.3286 1047.3210621.3207 768.6528 1397.551 287.9291 275.3006 924.40391674.535 2206.614 749.2892 1163.5029493.3522
 Calculating module eigengenes block-wise from all genes
   Flagging genes and samples with too many missing values...
    ..step 1
 ....pre-clustering genes to determine blocks..
   Projective K-means:
   ..k-means clustering..
   ..merging smaller clusters...
Block sizes:
gBlocks
   1    2    3 
4997 4015 1207 
 ..Working on block 1 .
    TOM calculation: adjacency..
    ..will use 4 parallel threads.
     Fraction of slow calculations: 0.000000
    ..connectivity..
    ..matrix multiplication (system BLAS)..
    ..normalization..
    ..done.
   ..saving TOM for block 1 into file norm_genes_TOM-block.1.RData
 ....clustering..
 ....detecting modules..
 ....calculating module eigengenes..
 ....checking kME in modules..
 ..Working on block 2 .
    TOM calculation: adjacency..
    ..will use 4 parallel threads.
     Fraction of slow calculations: 0.000000
    ..connectivity..
    ..matrix multiplication (system BLAS)..
    ..normalization..
    ..done.
   ..saving TOM for block 2 into file norm_genes_TOM-block.2.RData
 ....clustering..
 ....detecting modules..
 ....calculating module eigengenes..
 ....checking kME in modules..
 ..Working on block 3 .
    TOM calculation: adjacency..
    ..will use 4 parallel threads.
     Fraction of slow calculations: 0.000000
    ..connectivity..
    ..matrix multiplication (system BLAS)..
    ..normalization..
    ..done.
   ..saving TOM for block 3 into file norm_genes_TOM-block.3.RData
 ....clustering..
 ....detecting modules..
 No modules detected in block 3
  ..reassigning 1 genes from module 1 to modules with higher KME.
  ..reassigning 1 genes from module 9 to modules with higher KME.
  ..reassigning 3 genes from module 10 to modules with higher KME.
  ..reassigning 3 genes from module 11 to modules with higher KME.
  ..reassigning 1 genes from module 14 to modules with higher KME.
 ..merging modules that are too close..
     mergeCloseModules: Merging modules whose distance is less than 0.2
       Calculating new MEs...
   0    1    2    3    4    5    6    7    8    9   10   11 
8786  446  260  191   95   93   86   62   56   53   49   42 
11
  • 11 modules with between 42 and 446 genes
  • 8786 genes not assigned to a module
1433
1433

Network visualization

TOM calculation: adjacency..
..will use 4 parallel threads.
 Fraction of slow calculations: 0.000000
..connectivity..
..matrix multiplication (system BLAS)..
..normalization..
..done.
0610030E20Rik1600020E01RikZfp9741700094D03RikLrmda1810026B05RikNdufaf82010107E04Rik2210016L21Rik2310033P09RikZmpste24Zmym6Znhit2Znrd1Znrf1Znrf2Zrsr2Zscan26Zswim8Zzef1
0610030E20Rik11000100000000000100
1600020E01Rik11010100000100100110
Zfp97400100000000010000000
1700094D03Rik01010100000100000100
Lrmda00001000000000000000
1810026B05Rik11010100000100100110

Module analysis

446
[1] 1
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
3120  GO:0006325            1.382659e-17                        1         59
5992  GO:0016569            1.359305e-14                        1         48
4831  GO:0010467            2.437477e-13                        1        177
5993  GO:0016570            7.247069e-13                        1         40
9691  GO:0034641            1.068901e-12                        1        194
14183 GO:0051171            1.176385e-12                        1        156
      numInCat                                              term ontology
3120       676                            chromatin organization       BP
5992       483                            chromatin modification       BP
4831      4700                                   gene expression       BP
5993       380                              histone modification       BP
9691      5477      cellular nitrogen compound metabolic process       BP
14183     3764 regulation of nitrogen compound metabolic process       BP
[1] 2
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
9894  GO:0034976            3.199595e-08                1.0000000         16
3466  GO:0006810            7.546882e-08                1.0000000         94
5353  GO:0015031            9.893192e-08                1.0000000         50
14223 GO:0051234            2.388994e-07                1.0000000         95
12344 GO:0045184            4.804425e-07                0.9999999         52
13409 GO:0048193            9.212631e-07                0.9999998         14
      numInCat                                     term ontology
9894       201 response to endoplasmic reticulum stress       BP
3466      4060                                transport       BP
5353      1638                        protein transport       BP
14223     4209            establishment of localization       BP
12344     1798    establishment of protein localization       BP
13409      186                  Golgi vesicle transport       BP
[1] 3
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
3466  GO:0006810            3.075944e-24                        1         81
14223 GO:0051234            3.707310e-24                        1         82
14189 GO:0051179            8.161022e-24                        1         91
14937 GO:0055114            4.936305e-22                        1         43
12757 GO:0046034            4.491817e-20                        1         24
4464  GO:0009167            2.443053e-19                        1         24
      numInCat                                                  term ontology
3466      4060                                             transport       BP
14223     4209                         establishment of localization       BP
14189     5314                                          localization       BP
14937      878                           oxidation-reduction process       BP
12757      180                                 ATP metabolic process       BP
4464       205 purine ribonucleoside monophosphate metabolic process       BP
[1] 4
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] 5
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] 6
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] 7
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
1051  GO:0002376                       0                        1         39
1089  GO:0002443                       0                        1         12
4623  GO:0009615                       0                        1         14
9394  GO:0034097                       0                        1         16
14446 GO:0051607                       0                        1         12
16685 GO:0071345                       0                        1         15
      numInCat                                   term ontology
1051      2150                  immune system process       BP
1089       290            leukocyte mediated immunity       BP
4623       306                      response to virus       BP
9394       553                   response to cytokine       BP
14446      268              defense response to virus       BP
16685      458 cellular response to cytokine stimulus       BP
[1] 8
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
       category over_represented_pvalue under_represented_pvalue numDEInCat
15   GO:0000028                       0                        1          7
936  GO:0002181                       0                        1          9
3176 GO:0006412                       0                        1         40
3250 GO:0006518                       0                        1         40
3463 GO:0006807                       0                        1         46
3972 GO:0008152                       0                        1         46
     numInCat                                term ontology
15         19    ribosomal small subunit assembly       BP
936        49             cytoplasmic translation       BP
3176      597                         translation       BP
3250      738           peptide metabolic process       BP
3463     5797 nitrogen compound metabolic process       BP
3972     9802                   metabolic process       BP
[1] 9
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] 10
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] 11
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 3129 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
14458 GO:0051641            1.957635e-06                0.9999997         16
9673  GO:0034613            1.885418e-05                0.9999969         12
3938  GO:0008104            1.921928e-05                0.9999962         15
16390 GO:0070727            2.034254e-05                0.9999966         12
      numInCat                                term ontology
14458     2158               cellular localization       BP
9673      1444       cellular protein localization       BP
3938      2274                protein localization       BP
16390     1455 cellular macromolecule localization       BP
<!DOCTYPE html>
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[1] "10: 0 - 0%"
[1] "11: 0 - 0%"

Median of gene counts in the modules over ages

Module 1: chromatine/chromosome organization/RNA metabolic process

Module 2: response to endoplasmic reticulum stress/transport

Module 3: metabolic process (ATP, ribonucleoside)

Module 4

Module 5

Module 6: metabolic process (primary, cellular)

Module 7: immune system

Module 8: translation/rRNA

Module 9

Module 10: organelle

Module 11: localization